CN107255711A - Osteopontin is used for the purposes for preparing or screening acute-on-chronic liver failure diagnostic reagent - Google Patents
Osteopontin is used for the purposes for preparing or screening acute-on-chronic liver failure diagnostic reagent Download PDFInfo
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- CN107255711A CN107255711A CN201710326873.5A CN201710326873A CN107255711A CN 107255711 A CN107255711 A CN 107255711A CN 201710326873 A CN201710326873 A CN 201710326873A CN 107255711 A CN107255711 A CN 107255711A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
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Abstract
The invention belongs to biomedical and medical diagnosis on disease field, and in particular to osteopontin is used for the purposes for preparing or screening acute-on-chronic liver failure diagnostic reagent.Differentiation acute-on-chronic liver failure whether biomarker can be used as the invention firstly discloses osteopontin（Diagnosis effect is up to 0.9454）, hence it is evident that higher than M30（0.7167）、M65（0.8319）And M30/M65（0.8100）, and with clinically common counter MELD（0.9317）Almost maintain an equal level.
Description
Technical field
The invention belongs to biomedical and medical diagnosis on disease field, and in particular to osteopontin is used to preparing or screening slow urgent
The purposes of property hepatic failure diagnostic reagent.
Background technology
Osteopontin (Osteopontin, OPN) is a kind of multifunctional protein, is distributed widely in Various Tissues and cell,
Immune, angiogenesis, fiber generation and tumour row into etc. during play an important role.Osteopontin with it by matching somebody with somebody
Body integral protein, CD44, lipopolysaccharides, vimentin and MyD88 effects are sent out with during intercellular signal transduction in the cell
Important function is waved, so as to adjust cell propagation, migration, inflammation, fibrosis and canceration.Substantial amounts of research reports bone bridge at present
Albumen is sent out in the disease of acute liver damage, NASH, alcoholic liver, hepatitis, Hepatic fibrosis and cirrhosis and liver cancer
The signal that key is played during hair tonic exhibition goes to effect.
Acute-on-chronic liver failure (Acute-on-Chronic Liver Failure, ACLF) is on the basis of chronic liver disease
The acute hepatic decompensation of appearance.This is different from chronic liver function decompensation, and the latter refers on liver cirrhosis basis, liver function
Chronic liver function caused by progressive decline with ascites or portal hypertension, coagulation disorders and hepatic encephalopathy etc. mainly to show
Can decompensation.Slow urgent type hepatic failure is mainly shown as multiple organ failure, and the death rates on the 28th along with more than 30% and
More than 60% death rates on the 90th (Bernal W, Jalan R, Quaglia A, Simpson K, Wendon J, Burroughs
A. Acute-on-chronic liver failure.Lancet 2015.).Therefore, early stage occurs for acute-on-chronic liver failure
Diagnosis in time and treatment are most important for survival of patients.The diagnostic criteria of current acute-on-chronic liver failure is still disputable, CLIF-
C OF scorings are one of current Main Diagnosis standards.There is research by there is sub- massive necrosis region on pathological section from decompensation
Acute-on-chronic liver failure patient is distinguished in liver cirrhosis patient.But above diagnostic method or complicated and time consumption or invasive, for
Diagnosis has significant limitation in time.Thus, the new diagnosis that is sensitive, fast and efficiently aiding in acute-on-chronic liver failure of development
Or monitoring of diseases progress method, for clinic it is timely diagnosis and treat most important.
The content of the invention
In order to overcome defect of the prior art, it is used to preparing or screening slow it is an object of the invention to provide osteopontin
The purposes of extra urgaent dispatch hepatic failure diagnostic reagent.
Acute-on-chronic liver failure is being prepared another object of the present invention is to the reagent for providing specific recognition osteopontin
Purposes in diagnostic kit.
Another object of the present invention is to provide a kind of acute-on-chronic liver failure diagnostic kit.
Another object of the present invention is to provide osteopontin as the purposes of acute-on-chronic liver failure biomarker.
Another object of the present invention is to provide a kind of acute-on-chronic liver failure diagnostic method.
To achieve these goals and other related purposes, the present invention is to use following technical scheme：
The first aspect of the present invention is used for the use for preparing or screening acute-on-chronic liver failure diagnostic reagent there is provided osteopontin
On the way.
Preferably, the osteopontin is used as biomarker.
More excellent, the osteopontin is used as serum biomarkers.
Preferably, osteopontin is used to preparing or screening acute-on-chronic liver failure diagnostic reagent, including both sides content：
First, osteopontin is used to prepare acute-on-chronic liver failure diagnostic reagent, refer to osteopontin as slow urgent
Property hepatic failure diagnosis index be applied to the preparation of acute-on-chronic liver failure diagnostic reagent.
In some embodiments, can be using osteopontin as standard items or positive control, for the bone in sample serum
The detection of pontin protein level.
Second, osteopontin is used to screen acute-on-chronic liver failure diagnostic reagent, refer to osteopontin as slow urgent
Property hepatic failure identification target sieving specific recognition osteopontin reagent so that be used as acute-on-chronic liver failure diagnosis examination
Agent, to detect acute-on-chronic liver failure.
In some embodiments, the antibody of osteopontin is specifically bound based on described osteopontin screening or matched somebody with somebody
Body, so as to be used as acute-on-chronic liver failure diagnostic reagent.For example, osteopontin enzyme linked immunological kit (is purchased from R＆D Systems
Company, article No.：DOST00 the reagent in) can be with specific recognition osteopontin, therefore can be examined as acute-on-chronic liver failure
Disconnected reagent.
The second aspect of the present invention is examined preparing acute-on-chronic liver failure there is provided the reagent of specific recognition osteopontin
Purposes in disconnected kit.
In some embodiments, the reagent of the specific recognition osteopontin can be specific binding osteopontin
Antibody or part.The antibody can be monoclonal antibody or polyclonal antibody.
For example, osteopontin enzyme linked immunological kit (is purchased from R＆D Systems companies, article No.：DOST00 the reagent in)
Can be with specific recognition osteopontin, therefore box can be tried as acute-on-chronic liver failure diagnosis is prepared.
In the third aspect of the present invention there is provided a kind of acute-on-chronic liver failure diagnostic kit, in described kit
Reagent at least containing specific recognition osteopontin.
In some embodiments, the reagent of the specific recognition osteopontin can be specific binding osteopontin
Antibody or part.The antibody can be monoclonal antibody or polyclonal antibody.
In some embodiments, also contain in described kit：It is immune to combine (such as antigen-antibody combination) reagent；Or
Enzyme linked immunosorbent detection (ELISA) reagent.For example, osteopontin enzyme linked immunological kit (is purchased from R＆D Systems companies, article No.：
DOST00)。
In the fourth aspect of the present invention, there is provided purposes of the osteopontin as acute-on-chronic liver failure biomarker.
Preferably, the biomarker is serum biomarkers.
In the fifth aspect of the present invention, there is provided a kind of method for diagnosing acute-on-chronic liver failure, including detection sample blood
The level of osteopontin in clear.
Compared with prior art, the beneficial effects of the present invention are：
(it can be examined as differentiation acute-on-chronic liver failure whether biomarker the invention firstly discloses osteopontin
0.9454) disconnected effect is up to, hence it is evident that higher than M30 (0.7167), M65 (0.8319) and M30/M65 (0.8100), and with clinically
Common counter MELD (0.9317) almost maintains an equal level.
Brief description of the drawings
Fig. 1：Osteopontin is in acute-on-chronic liver failure patient, Decompensated liver cirrhosis patient, patients with hepatocellular carcinoma and normal
The detection of protein expression level in human serum.
Fig. 2：The egg of M30, M65 and M30/M65 in acute-on-chronic liver failure patient and Decompensated liver cirrhosis patients serum
The detection of white expression.
Fig. 3：Osteopontin, MELD, M30, M65 and M30/M65 work for diagnosing the subject of acute-on-chronic liver failure
The drafting of feature (ROC) curve.
Embodiment
The present invention has found to lose with Healthy People, patients with hepatocellular carcinoma and hepatic sclerosis first by in-depth study extensively
Compensatory patient is compared, and osteopontin content is significantly raised in acute-on-chronic liver failure peripheral blood in patients serum, with End-stage liver disease
Model (MELD), total bilirubin, creatinine level are proportionate, and related to disease and coincident with severity degree of condition.Osteopontin
Can as acute-on-chronic liver failure a kind of diagnosis marker, contribute to acute-on-chronic liver failure susceptibility analysis, diagnosis
And treatment.The medicine and diagnostic techniques designed according to human osteopontin, declines available for the slow extra urgaent dispatch liver for diagnosing and treating the mankind
Exhaust.The present invention is completed on this basis.
Osteopontin and application thereof
In the present invention in full, the osteopontin is known albumen, and English is Osteopontin, abbreviation OPN.People's bone bridge
The protein sequence of albumen refers to GenBank：BAA03554.1.
Among prior art, also not using osteopontin as the molecular labeling of acute-on-chronic liver failure relevant report.
The present invention is found through experiments that osteopontin expression quantity in acute-on-chronic liver failure patients serum is significantly higher than just
Ordinary person's group.Therefore, the invention provides a kind of purposes of osteopontin as acute-on-chronic liver failure mark.It is used as the present invention
Preferred embodiment, osteopontin as acute-on-chronic liver failure Virus monitory mark.Virus monitory convenient material drawing, operation letter
Singly, it is easy to check, patient is easy to receive, and assesses prognosis, guiding treatment.
Above-mentioned new discovery based on the present inventor, using mark of the osteopontin as diagnosis acute-on-chronic liver failure
Thing：(i) antidiastole of acute-on-chronic liver failure, and/or susceptible analysis are carried out；(ii) the slow extra urgaent dispatch of correlated crowd is assessed
Hepatic failure medicine, curative effect of medication, patient's prognosis, and select suitable treatment method；(iii) correlated crowd is assessed slowly to add
Acute hepatic failure risk, detect and carry out early prevention and treatment.
Therefore, the invention provides the purposes of osteopontin, for preparation or Screening Diagnosis (or detection) slow extra urgaent dispatch liver
The reagent or kit of exhaustion.
Detection reagent
Presence or absence and the expression of osteopontin, these technologies can be detected using various techniques known in the art
It is all contained in invention.For example, prior art such as Southern blottings, Wester blottings etc. can be used, these methods can be tied
Conjunction is used.
The present invention is also provided for detecting the presence or absence of osteopontin and the reagent of expression in analyte.It is excellent
Choosing, when carrying out the detection of protein level, the antibody of specific recognition osteopontin can be taken to determine depositing for osteopontin
Whether.
It is also using the antibody of osteopontin is specifically bound come the method for testing and analyzing osteopontin expression situation in thing
Technology well known in the art.
The antibody of bind osteopontin can be prepared by various technologies known to a person skilled in the art, can also be used
Commercialized antibody.For example, the osteopontin of purifying, can be applied to animal to induce polyclonal antibody to produce.Similarly
, the cell of expression osteopontin can be used to immune animal to produce antibody.The antibody of this discovery can also be monoclonal antibody.
Such clonal antibody can be prepared using hybridoma technology (see Kohler et al., Nature256；495,1975；Kohler etc.
People, Eur.J.Immunol.6:511,1976；Hammerling et al., In Monoclonal Antibodies and T
Cell Hybridomas, Elsevier, N.Y., 1981).
Animal, such as rabbit, mouse, rat can be immunized with osteopontin in the production of polyclonal antibody.A variety of adjuvants can use
In enhancing immune response, including but not limited to Freund's adjuvant etc..
It is also known to those skilled in the art that specific detectable signal is marked in monoclonal antibody or polyclonal antibody
Technology, for example, can be by horseradish root peroxidase conjugate in the antibody of described specific binding osteopontin, so as to pass through
Chromogenic reaction come learn antigen-antibody binding reaction generation whether.
Whether there is the method for osteopontin in a kind of detection testing sample, entered using the specific antibody of osteopontin
Row detection, it includes：Sample is contacted with Effect of Osteopontin-specific antibody；See whether to form antibody complex, form anti-
Nanocrystal composition, which is meant that, has osteopontin in sample.
Detection kit
Present invention also offers the presence or absence for detecting osteopontin in analyte with the kit of expression,
The kit includes：Specifically bind the antibody or part of osteopontin.
In addition, may also include in described kit immune with reference to (antigen-antibody combination) reagent or enzyme linked immunosorbent detection
(such as ELISA) reagent；And the various reagents needed for for hybridizing, developing the color etc., include but is not limited to：It is hybridization solution, enzyme, right
According to liquid, nitrite ion, cleaning solution etc..
Main advantages of the present invention are：
The present invention is disclosed by research and facing for acute-on-chronic liver failure is diagnosed and monitored using Serum osteopontin content
Bed purposes.It is of the invention that the method for detecting osteopontin content in peripheral blood is applied to diagnosis acute-on-chronic liver failure for the first time,
Especially find that content M30, M65, M30/M65 tri- that other people have found than early stage of osteopontin in detection peripheral blood is slow first
The serum mark of extra urgaent dispatch hepatic failure is sensitiveer, and then verifies a kind of method for detecting osteopontin content in peripheral blood, makees
To prepare the kit of diagnosis acute-on-chronic liver failure.This to clinical acute-on-chronic liver failure patient timely diagnosis and the state of an illness prison
Survey and provide more sensitive, efficient, stable approach.And these innovative discoveries of the present inventor, are that slow extra urgaent dispatch liver declines
The quick diagnosis and monitoring exhausted provides theoretical foundation, new detection method, has important to basic research and clinical practice
Meaning.
Illustrate embodiments of the present invention below by way of specific instantiation, those skilled in the art can be by this specification
Disclosed content understands the further advantage and effect of the present invention easily.The present invention can also pass through specific realities different in addition
The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints with application, without departing from
Various modifications or alterations are carried out under the spirit of the present invention.
Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, the protection domain being not intended to be limiting of the invention；In description of the invention and claims, unless in text
Explicitly point out in addition, singulative " one ", " one " and " this " include plural form.
When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range
Any one numerical value can select between point and two end points.Unless otherwise defined, in the present invention all technologies for using and
Scientific terminology is identical with the meaning that those skilled in the art of the present technique are generally understood that.Except the specific method used in embodiment, equipment,
Outside material, according to those skilled in the art to the grasp of prior art and the record of the present invention, it can also use and this
Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real
The existing present invention.
Unless otherwise indicated, disclosed in this invention experimental method, detection method, preparation method using this technology lead
Domain conventional molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of association area.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc.
MOLECμLAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLEC μ LAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLEC μ LAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Embodiment 1
1. experimental method
Collect 70 confirmation acute-on-chronic liver failure patients (ACLF+), 67 Decompensated liver cirrhosis patients (ACLF-), 50
Example patients with hepatocellular carcinoma (HCC), the peripheral blood sample of 30 normal persons (Health), each part Blood Sample Collection is in coagulation vessel
In, volume is 5ml or so, 5000rpm centrifugation 10min, draws the serum of upper strata clarification.Serum is detected by ELISA kit
Middle osteopontin, M30, M65 concentration (osteopontin enzyme linked immunological kit be purchased from R＆D Systems companies, article No.：
DOST00；M30, M65 enzyme linked immunological kit are purchased from VLVbio companies, and article No. is respectively nr.10011 and nr.10040),
MELD, total bilirubin and creatine concentration are obtained by patient history data.ROC is carried out using the softwares of GraphPad Prism 6.0
Drawing of Curve, is examined comparing group with Mann-Whitney, uses correlation analysis Spearman correlation tests.P<0.05
Think that there were significant differences.
2. experimental result and analysis
Relatively see Fig. 1 between the level and each group of osteopontin in serum.
As shown in Figure 1, osteopontin levels pole is significantly higher than other three groups of (P in acute-on-chronic liver failure patients serum<
0.0001), the level of osteopontin can decline as blood serum designated object for diagnosing and monitoring slow extra urgaent dispatch liver in prompting serum
Exhaust patient.
M30, M65 and M30/M65 compare in Decompensated liver cirrhosis group and acute-on-chronic liver failure group in serum sees Fig. 2.
As shown in Figure 2, M30, M65 are significantly higher than Decompensated liver cirrhosis group (P in acute-on-chronic liver failure patient<
0.0001), M30/M65 is substantially less than Decompensated liver cirrhosis group (P in acute-on-chronic liver failure patient<0.0001).This with before
Reported in literature be consistent.
Osteopontin and MELD, total bilirubin, the correlation detection of creatinine in acute-on-chronic liver failure patient, as a result such as
Shown in table 1.
Table 1
As shown in Table 1, osteopontin levels and its MELD, total bilirubin and creatinine in acute-on-chronic liver failure patients serum
Be proportionate (P<0.01), it is seen that osteopontin levels are relevant with disease severity in acute-on-chronic liver failure patients serum.
Osteopontin, MELD, M30, M65 and M30/M65 for diagnose acute-on-chronic liver failure Receiver Operating Characteristics
(ROC) drafting of curve is as shown in table 2：
Table 2
As shown in Table 2, acute-on-chronic liver failure patient is diagnosed to be from Decompensated liver cirrhosis patient, with bone bridge egg in serum
White level is as the diagnosis effect (0.9454) of biomarker apparently higher than M30 (0.7167), M65 (0.8319) and M30/
M65 (0.8100), and almost maintain an equal level with clinically common counter MELD (0.9317).
In summary, the invention provides one kind is in decompensation hepatic sclerosis quick diagnosis and screens acute-on-chronic liver failure trouble
The method of person, so as to realize the timely of patient and correct treatment；The present invention can be additionally used in the course advancement for monitoring patient, so as to
In adjustment therapeutic strategy, prevent treatment is incorrect not in time from causing death, great clinical value and application prospect.
Embodiment above is, in order to illustrate embodiment disclosed by the invention, can not to be interpreted as the limit to the present invention
System.In addition, method, the change of composition in various modifications listed herein and invention, are not departing from the scope of the present invention
Be obvious for those skilled in the art on the premise of spirit.Although having combined a variety of specific of the present invention
Preferred embodiment has carried out specific description to the present invention, it is to be understood that, the present invention should not be limited only to these specific embodiments.
In fact, various modifications obvious for those skilled in the art as described above should all include to obtain invention
Within the scope of the invention.
Claims (10)
1. osteopontin is used for the purposes for preparing or screening acute-on-chronic liver failure diagnostic reagent.
2. purposes according to claim 1, it is characterised in that the osteopontin is used as biomarker.
3. purposes according to claim 1, it is characterised in that the osteopontin is used as serum biomarkers.
4. purposes according to claim 1, it is characterised in that osteopontin is used to preparing or screening acute-on-chronic liver failure
Diagnostic reagent, including both sides content：First, osteopontin be used for prepare acute-on-chronic liver failure diagnostic reagent, refer to by
Osteopontin is applied to the preparation of acute-on-chronic liver failure diagnostic reagent as acute-on-chronic liver failure diagnosis index, can be by bone bridge
Albumen is as standard items or positive control, the detection for the osteopontin levels in sample serum；Second, osteopontin is used for
Acute-on-chronic liver failure diagnostic reagent is screened, is referred to the identification target sieving of osteopontin as acute-on-chronic liver failure is special
Property identification osteopontin reagent so that as acute-on-chronic liver failure diagnostic reagent, to detect acute-on-chronic liver failure.
5. purposes of the reagent of specific recognition osteopontin in acute-on-chronic liver failure diagnostic kit is prepared.
6. purposes according to claim 5, it is characterised in that the reagent of the specific recognition osteopontin is specificity
The antibody or part of bind osteopontin.
7. purposes according to claim 6, it is characterised in that the antibody is monoclonal antibody or polyclonal antibody.
8. osteopontin is used as the purposes of acute-on-chronic liver failure biomarker.
9. purposes according to claim 8, it is characterised in that the biomarker is serum biomarkers.
10. the level of osteopontin in a kind of method for diagnosing acute-on-chronic liver failure, including detection sample serum.
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| CN110824167A (en) * | 2019-07-03 | 2020-02-21 | 成都市第五人民医院(成都市老年病医院) | Application of marker for predicting prognosis of acute liver failure in preparation of prognosis evaluation kit for predicting prognosis of acute liver failure |
| CN113063937A (en) * | 2021-03-16 | 2021-07-02 | 首都医科大学附属北京佑安医院 | Evaluation markers for acute-on-chronic liver failure and their applications |
| CN113528639A (en) * | 2021-06-04 | 2021-10-22 | 南方医科大学南方医院 | Marker for predicting prognosis of chronic acute hepatic failure and application thereof |
| CN113650507A (en) * | 2021-08-25 | 2021-11-16 | 汤恩智能科技(常熟)有限公司 | Parking method and terminal for automatic walking vehicle |
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| CN110824167A (en) * | 2019-07-03 | 2020-02-21 | 成都市第五人民医院(成都市老年病医院) | Application of marker for predicting prognosis of acute liver failure in preparation of prognosis evaluation kit for predicting prognosis of acute liver failure |
| CN113063937A (en) * | 2021-03-16 | 2021-07-02 | 首都医科大学附属北京佑安医院 | Evaluation markers for acute-on-chronic liver failure and their applications |
| CN113528639A (en) * | 2021-06-04 | 2021-10-22 | 南方医科大学南方医院 | Marker for predicting prognosis of chronic acute hepatic failure and application thereof |
| CN113650507A (en) * | 2021-08-25 | 2021-11-16 | 汤恩智能科技(常熟)有限公司 | Parking method and terminal for automatic walking vehicle |
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| CN107255711B (en) | 2019-02-22 |
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